1) A heme-deficient guanylate cyclase purified from rat liver homogenate did not respond to nitric oxide (NO) or nitroprusside unless heme, 1 MuM, and activator or other proteins (Beta-globin, bovine serum albumin, or ovalbumin) were presented. NO-hemoglobin (NOHb) activated activity greater than 150-fold (specific activity 10.5 Mumol/min/mg protein). At maximal effective concentrations of activator or other proteins, responsiveness to NO heme increased markedly. With 1 MuM NO heme, maximal activities obtained with activator (3 Mug or 0.2 MuM), Beta-globin (5 MuM), or bovine serum albumin (15 MuM) were similar (4.7 Mumol/min/mg) to that with NOHb. With 1 MuM protoporphyrin IX, specific activity with Beta-globin was equivalent to that of Beta-globin plus NO heme. A complex of Beta-globin, activator, or bovine serum albumin with heme or protoporphyrin IX was isolated from gel filtration. Activation by the complex of Beta-globin-heme plus NO increased linearly with concentration of the complex (0.75-6 MuM) in the assay. The role of activator or other proteins is apparently to stabilize the enzyme, perhaps through interaction with a hydrophobic domain(s). 2) The increase in cGMP content caused by nitroprusside was approximately 6-fold higher in rat aorta than in rat vena cava. Responsiveness to nitroglycerin in both tissues dependent on duration of equilibration of the tissue prior to addition of the drug. After 30 min of equilibration, cGMP ratio in aorta to vena cava with nitrogelycerin was 4 to 1 when cGMP was determined in assay (1 min with aorta, 3 min with vena cava) with nitroglycerin. After 1.5 h or equilibration, the vena cava was no longer responsive to nitroglycerin but aorta still responded markedly.